EP1200116B1 - VERWENDUNG VON FVIIa ODER EINES GEWEBEFAKTORAGONISTEN ZUR REGULIERUNG DER GENEXPRESSION UND ZELLMIGRATION ODER CHEMOTAXIS - Google Patents

VERWENDUNG VON FVIIa ODER EINES GEWEBEFAKTORAGONISTEN ZUR REGULIERUNG DER GENEXPRESSION UND ZELLMIGRATION ODER CHEMOTAXIS Download PDF

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EP1200116B1
EP1200116B1 EP00943706A EP00943706A EP1200116B1 EP 1200116 B1 EP1200116 B1 EP 1200116B1 EP 00943706 A EP00943706 A EP 00943706A EP 00943706 A EP00943706 A EP 00943706A EP 1200116 B1 EP1200116 B1 EP 1200116B1
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fviia
cell
cells
tissue factor
pdgf
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EP1200116A2 (de
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Mirella Ezban
Lars Christian Petersen
Agneta Siegbahn
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Novo Nordisk Health Care AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to regulating cell migration or chemotaxis by contacting the cell with FVIIa or another TF agonist, and determining the migration of said cell.
  • the invention also relates to the use of FVIIa or another TF agonist, for the preparation of a medicament for regulation of cell migration in a patient.
  • the present invention relates to a method of detecting the activity of compounds, in particular drug candidates that interact with cell migration.
  • FVIIa tissue factor
  • TF tissue factor
  • the role of TF in blood coagulation has been extensively studied.
  • the involvement of FVIIa as a proteolytic enzyme in the blood coagulation cascade is believed to be confined to the extracellular leaflet of TF expressing cells.
  • An intracellular activity of FVIIa was first implied when the sequence of TF showed homology to the cytokine/ interferon- or heamatopoietic receptor superfamily.
  • the subclass I of the heamotopoietic receptor family includes receptors for growth hormone, prolactin, interleukins 1 to 7, granulocyte- macrophage colony stimulating factors, erythropoitin and thrombopoitin.
  • Subclass II includes TF and receptors for interferon a and b.
  • TF resemblance of TF to this class of receptors was further substantiated with the appearance of the crystal structure.
  • Characteristic of this class of cytokine receptors that includes receptors for interferon b and g and IL-10 is that their activation lead to rapid tyrosine phosphorylation of the receptors themselves, as well as a subset of intracellular proteins.
  • an array of mitogen-activated (Ser/Thr) kinases (MAPK) is activated.
  • Ser/Thr kinases mitogen-activated kinases
  • FVIIa is believed, in a TF dependent manner, to induce mobilisation of intracellular Ca 2+ through activation of phospholipase C.
  • the mechanism by which FVIIa activates phospholipase c is not known, but tyrosine kinase activation has specifically been ruled out.
  • TF may influence an array of important biological functions other than coagulation., such as angiogenesis, embryo vascularization and tumor metastasis. At present, however, it is unclear how TF contributes to these biological processes.
  • the extracellular domain of TF consists of two fibronectin-type III-like modules, as in the typical class II cytokine receptor extracellular domain, raising the possibility that TF may play a role in signal transduction, the primary function of cytokine receptor.
  • TF has a very short cytoplasmic domain (only 21 amino acid residues in length) and lacks membrane-proximal motifs that mediate binding of the non-receptor Janus kinases (Jaks) that are essential for cytokine receptor signaling. Nonetheless, several biochemical findings suggest a signal transduction function for TF. Analysis of the human TF protein sequence revealed a putative phosphorylation site in the cytoplasmic domain, which is conserved in mouse, rat and rabbit TF. Specific serine residues in the cytoplasmic tail of TF are phosphorylated in cells following stimulation with protein kinase C activator.
  • TF cytoplasmic tail is phosphorylated in vitro at multiple sites when incubated with lysates of U87-MG cells.
  • a potential role for the TF cytoplasmic domain in signal transduction is also indicated in studies that showed prometastatic function of TF is critically dependent on the TF cytoplasmic domain.
  • TF cytoplasmic domain is shown to interact with actin-binding protein 280 (ABP-280) and supports cell adhesion and migration through recruitment of ABP-280 to TF-mediated adhesion contacts.
  • TF has also been shown to participate certain types of cell signaling by serving as a cofactor for its physiological ligand FVIIa in an extracellular signaling by a proteolytic mechanism.
  • FVIIa physiological ligand FVIIa
  • binding of FVIIa to cell surface TF is shown to induce intracellular Ca 2+ oscillations in a number of TF expressing cells, transient phosphorylation of tyrosine in monocytes, activation of MAP kinase, alteration in gene expression in fibroblasts and enhanced expression of urokinase receptor in tumor cells.
  • Catalytically inactive FVIIa fails to induce many of the above signaling responses, from Ca 2+ oscillations to MAP kinase activation and gene reduction, and it appears that the catalytic activity of FVIIa may be required for at least some TF-FVIIa-mediated signal transduction. At present, not much is known about signaling pathway(s) that are induced by proteolytically active FVIIa and how the signals generated by FVIIa could contribute to angiogenesis and tumor metastasis.
  • the present invention relates to usage of FVII and/or FVIIa and/or another TF agonist for the preparation of the medicament for therapeutic treatment of pathological conditions that can be related to cell migration or treated by specific regulation of cell migration or chemotaxis.
  • the invention relates to the use of FVII and/or FVII and/or another TF agonist for the preparation of a medicament for therapeutic treatment of pathological conditions that can be related to the regulation of expression of at least one gene in a cell e.g. the Cyr61 gene.
  • the invention in another aspect relates to method for inducing or enhancing cell migration, comprising the step of contacting said cell with a tissue factor agonist
  • the tissue factor agonist is FVII or FVIIa.
  • the invention in another aspect relates to a method of reducing or inhibiting cell migration, comprising the step of contacting the cell with a tissue factor antagonist.
  • tissue factor antagonist is modified FVII.
  • the cell is a human cell expressing tissue factor, including fibroblasts, smooth muscle cells, tumour cells, haematopoietic cells and epithelial cells.
  • the modified factor VII is selected from factor VII modified with dansyl-Phe-Pro-Arg chloromethyl ketone, dansyl-Glu-Gly-Arg chloromethyl ketone; Dansyl-Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethylketone, dansyl-D-Phe-Pro-Arg chloromethyl ketone, Dansyl-D-Glu-Gly-Arg chloromethyl ketone, dansyl-D-Phe-Phe-Arg chloromethyl ketone and D-Phe-Phe-Arg chloromethyletone.
  • the invention in another aspect relates to the preparation of a medicament for inducing or enhancing wound healing in a patient, for administering to said patient an effective amount of a pharmaceutical composition comprising Factor VIIa or factor VII or another tissue factor agonist or a combination thereof.
  • the invention relates to regulating the expression of at least one gene in a cell, comprising the step of either contacting said cell with a tissue factor agonist or contacting said cell with a tissue factor antagonist.
  • the gene is a gene belonging to the CCN gene family.
  • the gene is selected from the group consisting of Cyr61, CTFG, dopamine D2 receptor, EST Incyte PD 395116 or P2U nucleotide receptor.
  • the gene is Cyr61 gene.
  • the regulation is inducing or enhancing expression.
  • FVII or FVIIa or another tissue factor agonist induces or enhances gene expression, e.g. when the gene is a gene belonging to the CCN gene family, or the gene is selected from the group consisting of Cyr61, CTFG, dopamine D2 receptor, EST Incyte PD 395116 or P2U nucleotide receptor.
  • FVII or FVIIa or another tissue factor agonist reduces or inhibits gene expression, e.g., when the gene is EST PD674714.
  • Diseased states which may be treated, are pathological conditions such as, for example, atherosclerosis or angiogenesis.
  • Other states that may be treated is, for example, healing of wounds including regeneration of vessel walls and treatment of burns, or inflammation, or the regulation of cell migration in vitro such as, for example, growing of tissue.
  • Fig. 1A and 1B Flow cytometric analysis of TF expression in fibroblasts (1A). The cells were stained with either a murine monoclonal fluoresceinisothiocyanate (FITC)-conjugated mouse anti IgG-antibody (unfilled area) that was used as negative control or a monoclonal FITC-conjugated anti-tissue factor (TF) antibody (filled area).
  • FITC monoclonal fluoresceinisothiocyanate
  • TF monoclonal FITC-conjugated anti-tissue factor
  • Fig. 2A Effects of FVIIa and FFR-FVIIa on PDGF-BB induced chemotaxis in human fibroblasts.
  • ⁇ show the chemotactic response of fibroblasts to different concentrations of PDGF-BB.
  • Fibroblasts incubated with 100 nM FVIIa (•) or 100 nM FFR-FVIIa ( ⁇ ) migrated towards different concentrations of PDGF-BB. Results are means and SEM of three separate experiments. P-values less than 0.05,* was considered statistically significant (Student's t test).
  • Fig. 3 A-D The influence of different concentrations of FVIIa or FFR-FVIIa on PDGF-BB induced chemotaxis in fibroblasts.
  • ⁇ show migration of fibroblasts to different concentrations of PDGF-BB.
  • Fig. 4A A mixture of three monoclonal antibodies to TF blocks the effects of FVIIa and FFR-FVIIa on PDGF-BB induced chemotaxis in fibroblasts.
  • ⁇ show migration towards PDGF-BB of fibroblasts without TF antibodies, ⁇ fibroblasts preincubated with TF antibodies and 100 nM FVIIa, and ⁇ fibroblasts preincubated with TF antibodies and 100 nM FFR-FVIIa. Results are mean and SEM of three separate experiments.
  • Fig. 5A and 5B The influence of FXa on the chemotactic response to PDGF-BB induced by FVIIa.
  • Fibroblasts were preincubated with 200 nM TAP ( fig.5 A) ( ⁇ ) or with 0.2-2 ⁇ M TAP ( fig.5B ) ( ⁇ ) and then with 100 nM FVIIa ( ⁇ ). TAP was present during the entire experiments.
  • Chemotaxis was induced by different concentrations of PDGF-BB (5A) or by 0.1 ng/ml PDGF-BB (5B). Results are mean and SD of two separate experiments.
  • Fig. 6A The influence of thrombin on the chemotactic response to PDGF-BB induced by FVIIa.
  • Fibroblasts were preincubated with 5 U/mL (final concentration) Hirudin and then with 100 nM FVIIa. Hirudin was present during the entire experiments. Chemotaxis was induced by different concentrations of PDGF-BB. ⁇ show cells incubated with Hirudin alone and • cells with Hirudin and FVIIa. Results are mean and SD of two separate experiments.
  • Fig. 7A Effect of inhibition of PI3'-kinase on chemotaxis in fibroblasts incubated with FVIIa.
  • Cells were preincubated with varying concentrations of LY294002 for 30 min at 37°C, and then with 100 nM FVIIa ( ⁇ ) or without FVIIa ( ⁇ ). The inhibitor was present throughout the chemotaxis assay.
  • Chemotaxis was induced by 0.1 ng/mL PDGF-BB. Results are mean and SD of two separate experiments.
  • Fig. 8A AND 8B Effect of inhibition of PLC on chemotaxis in fibroblasts incubated with FVIIa.
  • Cells were incubated with varying concentrations of U73122 (active PLC inhibitor) (8A) or U73343 (inactive control) (8B) for 30 min at 37°C before incubation with or without 100 nM FVIIa, and then assayed in the Boyden chamber to a concentration gradient of 0.1 ng/mL PDGF-BB.
  • the agents were present during the entire experiments.
  • ⁇ show cells with U73122 or U73343 alone, ⁇ cells with U73122 or U73343 and FVIIa. Results are mean and SD of two separate experiments.
  • Fig. 9 Release of inositol trisphosphate (IP 3 ) from fibroblasts stimulated with FVIIa, FFR-FVIIa alone or in combination with PDGF-BB.
  • IP 3 inositol trisphosphate
  • Cells were labelled over night with myo [ 3 H] inositol, incubated with or without 100 nM FVIIa or FFR-FVIIa in the absence or presence of 10 ng/mL or 100 ng/mL PDGF-BB. Cells were then analysed for release in IP 3 . Open bars show cells without FVIIa or FFR-FVIIa (control), hatched bars show cells with FFR-FVIIa, and black bars show cells incubated with FVIIa.
  • Fig. 10 Tyrosine phosphorylation of PLC- ⁇ 1 in response to PDGF-BB alone (control), FVIIa or FFR-FVIIa in combination with PDGF-BB.
  • Cells were incubated with 100 nM FVIIa or FFR-FVIIa for one hour, and then with or without PDGF-BB at indicated concentrations. Cells were lysed and tyrosine phosphorylation of PLC- ⁇ 1 detected as described in methods.
  • Fig. 1 Northern blot analysis confirming the data obtained with cDNA microarray assay.
  • Ten g of total RNA from the same RNA samples that were used to isolate poly (A) RNA to generate probes for hybridization of cDNA microarray) were patiented to Northern blot analysis and probed with 32 P-labeled Cyr61 (a partial length cDNA, obtained from Genomic Systems).
  • Panel B The hybridization signals are quantified with Phosphorlmager (Molecular Dynamics).
  • Fig. 2 and 2B Time-dependent factor VIIa-induced expression of Cyr61.
  • Quiescent monolayers of WI-38 cells were treated with factor VIIa (5 g/ml) (2A) or PDGF-BB (10 ng/ml) (2B) for varying time periods.
  • Total RNA (10 g) was patiented to Northern blot analysis and probed with radio labeled Cyr61. Ethidium bromide staining of 28S ribosomal RNA of the corresponding blot is shown in the bottom panel as RNA loading control.
  • Fig.3 Dose-dependent factor VIIa-induced expression of Cyr61.
  • Quiescent monolayers of WI-38 cells were treated with varying doses of factor VIIa, 0, 0.1, 0.5, 2.0 and 5.0 g/ml for 45 min.
  • Total RNA (10 g) was patiented to Northern blot analysis and probed with radiolabeled Cyr61. Ethidium bromide staining of 28S ribosomal RNA of the corresponding blot is shown in the bottom panel as RNA loading control.
  • Fig. 4 Factor VIIa catalytic activity is required for the induced expression of Cyr61.
  • Quiescent monolayers of WI-38 cells were treated with a control serum-free medium or serum-free medium cotaining factor VIIa (5 g/ml) or active-site inactivated factor VIIa (VIIai, 5 g/ml) for 45 min.
  • Total RNA (10 g) was patiented to Northern blot analysis and probed with radiolabeled Cyr61. Ethidium bromide staining of 28S ribosomal RNA of the corresponding blot is shown in the bottom panel as RNA loading control.
  • Fig. 5 Factor VIIa-induced expression of Cyr61 is not abolished by specific inhibitors of factor Xa and thrombin.
  • Quiescent monolayers of WI-38 cells were treated with control medium or the medium containing factor VIIa (5 g/ml; 100 nM for 45 min. Cells were preincubated with 200 nM recombinant TAP lane 3) or hirudin (lane 4) for 30 min before exposure to factor VIIa for 45 min.
  • Total RNA (10 g) was patiented to Nothern blot analysis and probed with radiolabeled Cyr61. Ethidium bromide staining of 28S ribosomal RNA of the corresponding blot is shown in the bottom panel as RNA loading control.
  • Fig. 6 Effect of actinomycin-D and cycloheximide on factor VIIa-induced Cyr61 mRNA steady-state levels.
  • Quiescent monolayers of WI-38 cells were preincubated with a control vehicle, actinomycin D (10 g/ml) or cycloheximide (10 g/ml) for 30 min before the cells were exposed to factor VIIa (5 g/ml) for 45 min.
  • Total RNA (10 g) was patiented to Northern blot analysis and probed with radiolabeled Cyr61. Ethidium bromide staining of 28S ribosomal RNA of the corresponding blot is shown in the bottom panel as RNA loading control.
  • Fig. 7 Factor VIIa induces the expression of CTGF.
  • Quiescent monolayers of WI-38 cells were treated with factor VIIa (5 g/ml) for varying time periods.
  • Total RNA (10 g) was patiented to Northern blot analysis and probed with radio labeled CTGF. Ethidium bromide staining of 28S ribosomal RNA of the corresponding blot is shown As RNA loading control.
  • the present invention relates to the use of FVII or FVIIa or another TF agonist for the manufacture of a pharmaceutical composition for inducing or enhancing cell migration.
  • the present invention relates to the use of FVII, FVIIa or another TF agonist for the manufacture of a pharmaceutical composition for inducing or enhancing wound healing or angiogenesis.
  • the cell migration is in a subject.
  • the present invention concerns a method for inducing or enhancing cell migration in a patient, which comprises administering an effective amount of FVII or FVIIa or another TF agonist to said patient.
  • the effective amount is a daily dosage from about 5 ⁇ g/kg/day to about 500 ⁇ g/kg/day.
  • the present invention provides a mechanism for an activity of FVII and/or FVIIa that relates to stimulation of cell migration.
  • a mechanism provides the basis for establishing the involvement of FVII and/or FVIIa in pathological conditions in which TF expressing cells like endothelial cells, epithelial cells, fibroblasts, smooth muscle cells and monocytes/macrophages participate.
  • the invention furthermore provides the basis for identifying specific pharmacological targets that are useful for therapeutic intervention.
  • the present invention relates to usage of FVII and/or FVIIa in therapeutic treatment of pathological conditions that can be related to cell migration or treated by specific regulation of cell migration.
  • the present invention relates to a method of detecting drug candidates that regulate cell migration, which method comprise
  • the blood components which participate in what has been referred to as the coagulation "cascade” are proenzymes or zymogens, enzymatically inactive proteins, which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor.
  • Coagulation factors that have undergone such a conversion and generally referred to as “active factors”, and are designated by the addition of the letter "a" to the name of the coagulation factor (e.g. factor VIIa).
  • zinc-chelator is intended to comprise a compound that binds to factor VIIa and induces replacement of calcium ions with zinc ions within factor VIIa, thereby inhibiting the activity of factor VIIa or tissue factor-factor VIIa complex (TF-FVIIa).
  • a TF antagonist as defined according to the invention may be a zinc-chelating compound, e.g. a dihydroxamate or a dihydrazide with the hydroxamate or hydrazide groups located relative to each other in such a position that they are able to chelate a zinc ion.
  • the zinc-chelating compound acts in combination with FVIIa.
  • Zn 2+ -ions exert their inhibitory action in competition with a stimulatory effect of Ca 2+ -ions. It is predicted that Zn 2+ -ions displace Ca 2+ -ions from one or more calcium binding site(s) within FVIIa.
  • Zinc-chelating compounds e.g.
  • hydroxamates and hydrazides are capable of acting as powerfull supporters for binding of zinc ions in competition with calcium ions. Specific compounds thereby potentiate zinc inhibition of the activity of the factor VIIa/tissue factor complex.
  • the activity of factor VIIa in complex with tissue factor can be inhibited by a mechanism in which a zinc chelator binds to factor VIIa and facilitates replacement of Ca 2+ with Zn 2+ . By this action the chelator exerts a modulatory effect on TF at the normal concentration of free Ca 2+ and Zn 2+ ions in the blood.
  • FVII or Fact VII means “single chain” (zymogenic) coagulation factor VII.
  • Factor VIIa or “FVIIa” means "two chain” activated coagulation factor VII cleaved by specific cleavage at the Arg152-Ile153 peptide bond.
  • FVII and FVIIa may be purified from blood or produced by recombinant means. It is evident that the practice of the methods described herein is independent of how the purified factor VIIa is derived and, therefore, the present invention is contemplated to cover use of any factor VII or FVIIa preparations suitable for use herein. Preferred are human FVIIa.
  • FVII or FVIIa is also intended to include FVII variants wherein one or more amino acid residues has (have) been replaced.
  • modified factor VII is intended to mean FVIIa having at least one modification in its catalytic centre, which modification substantially inhibits the ability of modified FVIIa to activate FX and FIX.
  • modification includes amino acid substitution (or replacement) of one or more of the catalytic triad residues Ser344, Asp142 and His193, and also includes modification of catalytic triad residues with serine protease inhibitors such as organo-phosphor compounds, sulfanylfluoride, peptide halomethyl ketone or azapeptide.
  • FFR-FVIIa is one example of a FVIIai derivative obtained by blocking of the active centre of FVIIa with the irreversible inhibitor, D-phenylalanine-L-phenylaianine-L-argininine chloromethyl ketone (FFR cmk).
  • FVIIai derivates are inactivated FVIIa obtained or obtainable by blocking the active centre with L-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone, dansyl-L-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone, or dansyl-D-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone, Preferred is FFR-FVIIa (FVIIa inactivated by FFR cmk).
  • protein kinase is intended to indicate an enzyme that is capable of phosphorylating serine and/or threonine and/or tyrosine in peptides and/or proteins.
  • drug candidate is intended to indicate any sample, which has a biological function or exerts a biological effect in a cellular system.
  • the sample may be a sample of a biological material such as a microbial or plant extract, or it may be a sample containing a compound or mixture of compounds prepared by organic synthesis or genetic techniques.
  • TF agonist comprises compounds inducing
  • TF antagonist comprises
  • pharmacological targets is intended to indicate a protein that can alter the migration of TF expressing cells.
  • reporter gene is intended to indicate a DNA construct that, when transcribed, produces a protein that can be detected.
  • SRE promoter element means a DNA sequence that binds transcription factors induced by components present in serum.
  • TF expressing cell means any mammalian cell that expresses TF.
  • protein phosphorylation is intended to indicate phosphorylation of serine and/or threonine and/or tyrosine in peptides and/or proteins.
  • Modulation or regulation of cell migration is defined as the capacity of FVIIa or another TF agonist, or FVIIai or another TF antagonist to 1) either increase or decrease ongoing, normal or abnormal, cell migration, 2) initiate normal cell migration, and 3) initiate abnormal cell migration.
  • Modulation or regulation of gene expression is defined as the capacity of FVIIa or another TF agonist, or FVIIai or another TF antagonist to 1) either increase or decrease ongoing, normal or abnormal, cell migration, 2) initiate normal cell migration, and 3) initiate abnormal cell migration.
  • treatment is meant to include both prevention of an adverse condition and regulation of an already occurring condition with the purpose of inhibiting or minimising the condition.
  • Prophylactic administration of FVIIa or another TF agonist, or FVIIai or another TF antagonist is thus included in the term "treatment”.
  • one unit is defined as the amount of factor VII present in 1 ml of normal plasma, corresponding to about 0.5 ⁇ g protein. After activation 50 units correspond to about 1 ⁇ g protein.
  • patient is defined as any animal, in particular mammals, such as humans.
  • subject is used interchangeably with “patient”
  • Tissue factor is the cellular receptor for factor FVIIa (FVIIa) and the complex is principal initiator of blood coagulation.
  • FVIIa factor FVIIa
  • FVIIa factor FVIIa
  • Anti-TF antibodies inhibited the increase in chemotaxis induced by FVIIa/TF.
  • FFR-FVIIa active site-inhibited FVIIa
  • FVIIa induced the production of inositol-1,4,5-trisphosphate to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB were additive.
  • FFR-FVIIa did not induce any release of inositol-1,4,5,-trisphosphate.
  • the cellular migration response to PDGF-BB and FVIIa was totally blocked by a PLC-inhibitor, suggesting that activation of PLC is important for the response.
  • binding of FVIIa to TF can independent of coagulation, modulate cellular responses, such as chemotaxis, and the catalytic activity of FVIIa is necessary.
  • TF is believed to exert a function in tumour cell metastasis, but the mechanism is yet not known.
  • Ott et al. very recently identified actin-binding protein 280 (ABP-280) as a ligand for the TF cytoplasmic domain, providing a molecular pathway by which TF may support tumor cell metastasis.
  • ABSP-280 actin-binding protein 280
  • Human fibroblasts have a constitutive expression of TF. These cells also express receptors for platelet-derived growth factor (PDGF). PDGF induces in its target cells mitogenicity, actin reorganization and directed cell migration (chemotaxis).
  • PDGF-BB platelet-derived growth factor
  • chemotaxis directed cell migration
  • PI3'-kinase phosphatidylinositol 3'-kinase
  • PLC phospholipase C
  • IP 3 inositol-1,4,5-trisphosphate
  • TF is constitutively expressed on the plasma membrane of many extravascular cells, such as stromal fibroblasts in vascular adventitia and in fibrous capsules of liver, spleen and kidney.
  • extravascular cells such as stromal fibroblasts in vascular adventitia and in fibrous capsules of liver, spleen and kidney.
  • stromal fibroblasts in vascular adventitia and in fibrous capsules of liver, spleen and kidney.
  • TF can, however, be induced in monocytes/macrophages, vascular smooth muscle cells, endothelial cells and in a number of tumour cells by a variety of agents, including cytokines and growth factors. Induction at the transcriptional level occurs rapidly after stimulation, identifying TF as a growth-related immediate early gene.
  • the enhanced phosphorylation of PLC- ⁇ 1 correlated with a threefold higher IP 3 production compared to wild-type PDGF ⁇ -expressing cells.
  • the combination of FVIIa/TF and PDGF-BB induced about twofold increase in IP 3 production in human fibroblasts.
  • FVIIa/TF-induced IP 3 production did not correlate with phosphorylation of PLC- ⁇ 1.
  • Tyrosine phosphorylation of PLC- ⁇ 2 induced by FVIIa/TF cannot be excluded, but seems unlikely since the expression of PLC- ⁇ 2 is very low in human fibroblasts.
  • the intracellular part of TF is not endowed with intrinsic protein tyrosine kinase activity.
  • TF was found to be in close contact with actin and actin filament-binding proteins, such as ⁇ -actinin and ABP280 in lamellipodia and ruffled membrane areas in spreading epithelial cells.
  • actin and actin filament-binding proteins such as ⁇ -actinin and ABP280 in lamellipodia and ruffled membrane areas in spreading epithelial cells.
  • ABP 280 a member of the filamin subfamily, is required for normal function of lamellipodia and thus highly important for cell motility.
  • PI3'-kinase and PLC isozymes are implicated in chemotactic responses, such as mobilisation of actin-binding proteins.
  • Chemotaxis plays a pivotal role in wound healing, angiogenesis and metastasis. Chemotaxis is also an important component in the development of atherosclerotic plaques. In these processes a variety of cells express TF as well as PDGF and PDGF receptors. Restenosis is a major complication following interventional procedure of obstructed arteries. PDGF has been implicated in the vessel wall's response (neointima formation) to mechanical injury by mediating the migration and proliferation of smooth muscle cells and fibroblasts. We have shown now for the first time that FVIIa binding to TF-expressing cells have an increased chemotactic response to PDGF, which is independent of the coagulation.
  • fibroblasts are programmed to interpret the abrupt exposure to serum nor as a general mitogenic stimulus but as a specific physiological signal. Characterization of transcriptional activation in response to serum and growth factors also suggest that fibroblasts are an active participant in a conversation among the diverse cells which collectively control inflammation, angiogenesis and wound healing.
  • Cyr61 is an immediate-early gene that is transcriptionally activated by serum growth factors in fibroblasts. It encodes a secreted 40 kDa, cysteine-rich and heparin-binding protein that associates with extracellular matrix and cell surfaces. Cyr61 is a member of an emerging gene family of conserved and modular proteins characterized by the presence of an N-terminal secretory signal, followed by four modular structural domains and 38 cysteine residues that are largely conserved among members of the family.
  • the protein family now consists of six distinct members, including C yr61 , connective tissue growth factor ( C TGF) and an avian proto-oncoprotein, N ov (thus named as CCN family) (The CCN family is further described in Lau et al., Exp. Cell Res 248: 44-57, 1999 ).
  • Cyr61 protein is shown to (i) promote the attachment and spreading of endothelial cells in a manner similar to that of fibronectin, (ii) enhance the effects of bFGF and PDGF on the rate of DNA synthesis of fibroblasts and vascular endothelial cells (iii) promotes cell migration in both fibroblasts and endothelial cells.
  • Cyr61 acts as a ligand to integrin ⁇ ⁇ ⁇ 3 , an adhesion receptor known to be involved in signaling that regulates a number of cellular processes including angiogenesis and tumor metastasis.
  • Purified Cyr61 protein was shown to stimulate directed migration of human microvascular endothelial cell in culture through a ⁇ ⁇ ⁇ 3 -dependent pathway and induce neovascularization in rat corneas.
  • expression of Cyr61 in tumor cells promotes tumor growth and vascularization.
  • CTGF behaves very similar to Cyr61, subtle differences exist between them. For example, (a) CTGF has shown to be mitogenic in itself whereas Cyr61 has no intrinsic mitogenic activity but augments growth factor-induced DNA synthesis (b) Cyr61 stimulates chemotaxis whereas CTGF stimulates both chemotaxis and chemokinesis (c) although both Cyr61 and CTGF are ECM-associated signalling molecules, CTGF is shown to secrete into culture medium. Thus, it is possible that FVIIa regulates cellular functions locally via Cyr61 whereas acts at a distance from its site through the secretion of CTGF.
  • the regimen for any patient to be treated with FVIIa or another TF agonist or FVIIai or another TF antagonist as mentioned herein should be determined by those skilled in the art.
  • the daily dose to be administered in therapy can be determined by a physician and will depend on the particular compound employed, on the route of administration and on the weight and the condition of the patient.
  • An effective amount is suitably a daily dosage from about 5 ⁇ g/kg/day to about 500 ⁇ g/kg/day, preferably from about 10 ⁇ g/kg/day to 300 ⁇ g/kg/day, more preferred from about 15 ⁇ g/kg/day to 200 ⁇ g/kg/day, most preferred from about 20 ⁇ g/kg/day to 100 ⁇ g/kg/day.
  • the FVIIa or another TF agonist or FVIIai or another TF antagonist should be administered in one single dose, but it can also be given in multiple doses preferably with intervals of 4-6-12 hours depending on the dose given and the condition of the patient.
  • the FVIIa or another TF agonist or FVIIai or another TF antagonist may be administered intravenously or it may be administered by continuous or pulsatile infusion or it may be administered directly to the relevant site such as, for example, injected directly into a tumour.
  • FVIIa or another TF agonist or FVIIai or another TF antagonist is preferably administered by intraveneous injections and in an amount of about 100-100,000 units per kg body weight, and preferably in an amount of about 250 - 25,000 units per kg body weight corresponding to about 5-500 ⁇ g/kg, a dose that may have to be repeated 2-4 times per 24 hours.
  • compositions used according to this invention are prepared by methods known per se by the skilled artisan.
  • compositions suitable for use according to the present invention is made by mixing FVII, FVIIa or another TF agonist, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent.
  • suitable physiological acceptable carriers or diluents include sterile water and saline.
  • Suitable adjuvants include calcium, proteins (e.g. albumins), or other inert peptides (e.g. glycylglycine) or amino acids (e.g. glycine, or histidine) to stabilise the purified factor VIIa.
  • Other physiological acceptable adjuvants are non-reducing sugars, polyalcohols (e.g.
  • the adjuvants are generally present in a concentration of from 0.001 to 4% w/v.
  • the pharmaceutical preparation may also contain protease inhibitors, e.g. apronitin, and preserving agents.
  • the preparations may be sterilised by, for example, filtration through a bacteria-retaining filter, by incorporating sterilising agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile medium suitable for injection prior to or immediately before use.
  • Human purified factor VIIa suitable for use in the present invention is preferably made by DNA recombinant technology, e.g. as described by Hagen et al., Proc.Natl.Acad.Sci. USA 83: 2412-2416, 1986 or as described in European Patent No. 200.421 (ZymoGenetics).
  • Factor VIIa produced by recombinant technology may be authentic factor VIIa or a more or less modified factor VIIa provided that such factor VIIa has substantially the same biological activity for blood coagulation as authentic factor VIIa.
  • modified factor VIIa may be produced by modifying the nucleic acid sequence encoding factor VII either by altering the amino acid codons or by removal of some of the amino acid codons in the nucleic acid encoding the natural FVII by known means, e.g. by site-specific mutagenesis.
  • Factor VII may also be produced by the methods described by Broze and Majerus, J.Biol.Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J.Clin.Invest. 71: 1836-1841, 1983 . These methods yield factor VII without detectable amounts of other blood coagulation factors. An even further purified factor VII preparation may be obtained by including an additional gel filtration as the final purification step. Factor VII is then converted into activated FVIIa by known means, e.g. by several different plasma proteins, such as factor XIIa, IX a or Xa. Alternatively, as described by Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565 ), factor VII may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia fine Chemicals) or the like.
  • Mono Q® Phharmacia fine Chemicals
  • Modified factor VII suitable for use in the present invention is made, e.g. as described in International Publications Nos. 92/15686 , 94/27631 , 96/12800 and 97/47651 ZymoGenetics/Novo Nordisk).
  • Fibroblasts expressing active TF were incubated with 100 nM of FVIIa and seeded in the upper part of the modified Boyden chamber; while media containing 10% FBS and PDGF-BB at different concentrations were added below the 150 ⁇ m micropore filter.
  • the migration of the cells under conditions where medium containing 10% FBS without PDGF-BB was added below the filter was used as a measure of random migration, and calculated as 100% migration.
  • a significant migration response was recorded at a concentration of 0.01 ng/ml PDGF-BB in cells stimulated by FVIIa compared to 1 ng/ml PDGF-BB for cells not ligated with FVIIa, i.e.
  • the hyperchemotactic response is not mediated by FXa or by thrombin
  • FVIIa-induced signal transduction leading to the hyperchemotactic response to PDGF-BB was dependent on the catalytic activity of FVIIa it was important to determine whether signalling occurred directly or via FXa or thrombin generated by the FVIIa/TF complex.
  • the enhanced migration response transduced by FVIIa/TF was not blocked by 0.2-10 ⁇ M Tick anticoagulant peptide (TAP), which specifically blocks the active site of FXa and prevents a further activation of the coagulation cascade leading to thrombin formation ( Fig. 5A,5B ).
  • the hyperchemotactic response to PDGF-BB is influenced by PLC-dependent pathways, but independent of PI3'-kinase.
  • LY294002 a specific PI3'-kinase inhibitor, was able to block the chemotactic response induced by FVIIa/TF signalling.
  • Fibroblasts were pretreated with LY294002 at indicated concentrations for 30 minutes at 37°C before the addition of 100 nM FVIIa and assayed in the Boyden chamber as described.
  • the concentration of PDGF-BB was kept constant at 0.1 ng/ml throughout the assay, i.e. a very low concentration at which FVIIa/TF induced a significant chemotactic response.
  • LY294002 was present during the entire experiments.
  • Fig.7A shows that the migration response to PDGF-BB mediated by FVIIa/TF-signalling was unaffected by the inhibition of PI3'-kinase.
  • tyrosine phosphorylation of PLC- ⁇ 1 was studied. Fibroblasts were incubated in the absence or presence of 100 nM FVIIa or FFR-FVIIa for one hour, followed by the stimulation with 0, 2, 10 or 100 ng/ml PDGF-BB. After 5 minutes of incubation, the cells were lysed and PLC- ⁇ 1 was immunoprecipitated, separated by SDS-PAGE and immunoblotted with antiphosphotyrosine antibodies.
  • Human foreskin fibroblasts, AG1518 and AG1523 were grown to confluence in Eagle's MEM supplemented with 10% fetal bovine serum (FBS). Before use, the cells were detached by trypsinization (2.5 mg/ml for 10 min at 37°C), washed in Hank's balanced salt solution, and resuspended in Eagle's MEM with 10% FBS or in Ham's medium supplemented with 0.1 % FBS.
  • FBS fetal bovine serum
  • FVIIa Human FVIIa (Novo Nordisk A/S, Gentofte, Denmark), was expressed and purified as described 29 .
  • FFR-FVIIa Novo Nordisk
  • FFR-FVIIa Novo Nordisk
  • Tick anticoagulant peptide TMP
  • LY294002 U73122 and U73343 were obtained from Biomol (Plymouth Meeting, PA).
  • Anti-TF monoclonal antibodies TF8-5G9, TF9-5B7 and MTFH-1 ( Morrissey, J.H., Fair, D.S., Edgington, T.S. Monoclonal antibody analysis of purified and cell-associated tissue factor. Thromb. Res. 52, 247-261 (1988 )) was a kind gift of Dr. James H. Morrissey, Oklahoma Medical Research Foundation. The phosphotyrosine antibody, PY99 was from Santa Cruz, California.
  • Flow cytometry The surface expression of TF was analysed by immunofluorescence with a flow cytometer (Coulter Epics XL-MCL, Beckman Coulter, Fullerton, CA, Coulter Electronics, USA). The instrument was calibrated daily with Immuno-Check TM or Flow Check TM calibration beads (Coulter). For indirect immunofluorescence experiments AG1518 or AG1523 fibroblasts were washed twice with PBS containing 0.1% bovine serum albumin (BSA), incubated for 30 minut-ps on ice with a fluorescein-isothiocyanate (FITC)-labelled anti-human TF monoclonal antibody (4508CJ, American Diagnostica, Greenwich, Ct. USA). The anti-Aspergillus niger glucose oxidase monoclonal IgG1 (Dakopatts) was used as a negative control. Mean channel fluorescence intensity (MFI) and percentage of positive cells were determined for each sample.
  • MFI Mean channel fluor
  • TF activity The procoagulant activity of TF was determined as described by Lindmark et al. ( Lindmark, E., Tenno, T., Chen, J., Siegbahn, A. IL-10 inhibits LPS-induced human monocyte tissue factor expression in whole blood. Br. J. Haematol. 102, 597-604 (1998 )). Briefly, aliquots containing 0.2 x 10 5 AG1518 or AG1523 fibroblasts were washed twice with PBS, placed in the wells of a 96-well microtitreplate (Nunc, Roskilde, Denmark).
  • the procoagulant activity was measured in a two-stage amidolytic assay where a chromogenic substrate, S-2222 (Chromogenix, Mölndal, Sweden), is cleaved by FXa, which in turn is activated from FX by the TF/FVIIa complex.
  • Chemotaxis assay The migration response of fibroblasts was assayed by means of the leading front technique in a modified Boyden chamber, as previously described ( Siegbahn, A., Hanimacher, A., Westermark, B., Heldin, C-H. Differential effects of the various isoforms of platelet-derived growth factor on chemotaxis of flbroblasts, monocytes, and granulocytes. J. Clin. Invest. 85, 916-920 (1990 ) and Nistér, M., Hammacher, A., Mellström, K., Siegbahn, A., Rönnstrand, L., Westermark, B., Heldin, C-H.
  • a glioma-derived PDGF A chain homodimer has different functional activities from a PDGF AB heterodimer purified from human platelets.
  • Micropore filters pore size 8 ⁇ m were coated with a solution of type-1 collagen at room temperature over night. The filters were air dried for 30 minutes immediately before use.
  • PDGF-BB was diluted in assay media (Eagle's MEM with 10% FBS) and added below the filter in the chamber. The cells were incubated for 6 hours at 37°C in a humidified chamber containing 95% air/5% CO 2 . FVIIa or FFR-FVIIa were present during the entire experiment. The filters were then removed, fixed in ethanol, stained with Mayer's Hemalun, and mounted. Migration was measured as the distance of the two furthest migrating fibroblast nuclei of one high-power field (12.5x 24) in focus.
  • the migration distance in each filter was calculated as the mean of the readings of at least three different parts of the filter. Experiments were performed with two to four separate filters for each concentration of chemoattractant. For each set of experiments, the migration of fibroblasts toward the assay media served as control.
  • IP 3 Assay for release of inositol trisphosphate (IP 3 ).
  • PDGF-BB (0, 10 or 100 ng/ml) was added and the incubation was continued for 10 minutes at 37°C.
  • the IP 3 assay was performed as previously described by Eriksson et al. ( Eriksson, A., N ⁇ nberg, E., Rönnstrand, L., Engström, U., Hellman, U., Rupp, E., Carpenter, G., Heldin, C-H., Claesson-Welsh, L. Demonstration of functionally different interactions between phospholipase C- ⁇ and the two types of platelet-derived growth factor receptors. J. Biol. Chem. 270, 7773-7781 (1995 )).
  • Recombinant human VIIa a gift from Novo Nordisk (Gentofte, Denmark), was reconstituted in sterile water at a concentration of I to 1.3 mg/ml. The stock VIIa solutions were checked for contaminating trace levels of endotoxin using limulus amebocyte lysate (Bio Whittaker) and none was detected (detection level 30 pg).
  • Recombinant tick anticoagulant protein (TAP) was kindly provided by George Vlasuk (Corvas, San Diego, CA) and recombinant hirudin was obtained from either Sigma (St.Louis, MO) or Calbiochem (San Diego, CA). Purified human factor Xa and thrombin were, obtained from Enzyme Research Laboratories (Southbend, IN).
  • cDNA microarray WI-38 cells were cultured to 80% confluency and serum deprived for 24 hours to enter quiescent state as described above. The culture medium was replaced with fresh serum-free DMEM (supplemented with 5 mM CaCl 2 ) and allowed to stabilize for 2 h in culture incubator. Then, the cells were treated with purified recombinant VIIa (5 ⁇ g/ml) for 90 min. At the end of 90 min treatment, total RNA was isolated from untreated (control) and VIIa-treated cells using Trizol (GIBCO BRL). Poly (A) RNA was purified by a double pass over Oligo Tex mRNA isolation columns as described in manufacturer's technical bulletin (Qiagen). Eight hundred ng (800 ng) of highly purified poly (A) RNA from the control and VIIa-treated cells were sent for cDNA microarray analysis service (Human UniGEM V microarray, Genome Systems Inc, St. Louis, MO).
  • Nothern blots were prehybridized at 42°C with a solution containing 50% formamide, 5 x SSC, 50 mM Tris.HCl, pH 7.5, 0.1% sodium pyrophosphate, I% SDS, I% olyvinylpyrrolidone, I% Ficoll, 25 mM EDTA, 100 ⁇ g/ml denatured salmon sperm DNA and 1% BSA and hybridized with 32 P-labeled Cyr61 cDNA probe (106 cpm/ml). The hybridized membranes were exposed to either Dupont NEF or Fuji RX X-ray film.
  • the membranes were exposed to phosphor screen for I to 4 h, and the exposed screens were analyzed in a Phosphorlmger (Molecular Dynamics) using "Image-quant" software. To obtain mean values, the units (counts) obtained from different experiments were normalized to an internal control (counts present in control-treated sample).
  • the control plate in which known concentrations of reference cDNA was spiked into the probe generation reaction to measure sensitivity and monitor the reverse transcription reaction, purification determine hybridization efficiency and overall view of the quality and performance of the assay indicated the success of hybridization process.
  • Global analysis of experimental data revealed minimal differences in hybridization signals between the control and VII-treated samples- Only a small number of genes showed moderate differential expression.
  • the identity of the 3.5-fold upregulated gene was not revealed due to the proprietary nature.
  • VIIa- upregulated genes are Cyr61 (2.5-fold), dopamine D2 receptor (2.2-fold), EST Incyte PD 395116 (2-fold) and P2U nucleotide receptor (2-fold). It is interesting to note that CTGF, a gene belonging to the Cyr61 family, was I.8-fold higher in VIIa-treated cells compared to control cells. The downregulated transcript in VIIa-treated cells was EST PD674714 . We selected Cyr61 for further analysis.
  • Cyr61 Since it had been reported that expression of Cyr61 in mouse fibroblasts after stimulation with serum and growth factor was sustained for several hours (up to 8 to 10 h) before repression occurs, we have examined the effect of serum and PDGF on kinetics of Cyr61 expression in quiescent human fibroblasts, WI-38. As shown in Fig. 2B , Cyr61 is expressed only transiently upon stimulation with PDGF and become fully repressed 2 h after the addition of stimuli. Similar results obtained with serum-induced expression of Cyr61 (data not shown).
  • Factor VIIa-dose dependent induced expression of Cyr61 To determine dose-dependency of VIIa, quiescent fibroblasts were treated with varying doses FVIIa (0.1 to 5 ⁇ g/ml) for 45 min and then total RNA samples from the cells were patiented to Northern blot analysis. As shown in Fig. 3 , treatment of fibroblasts with as low as 0.1 ⁇ g/ml FVIIa was sufficient to induce the expression of Cyr61 and a plasma concentration of FVII(a) (0.5 ⁇ g/ml, 10 nM resulted in a prominent response, close to the maximal.
  • Factor VIIa-catalytic activity is required for Cyr61 induction.
  • WI-38 cells were treated with VIIa and active-site inactivated FVIIa (FVIIai) for 45 min and the expression of Cyr61 was evaluated by Northern blot analysis.
  • FVIIai failed to induce the expression of Cyr61 suggesting the requirement of FVIIa proteolytic activity.
  • FVIIai was shown to bind cell surface TF with the same or higher affinity than FVIIa.

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Claims (11)

  1. Verwendung eines Gewebefaktoragonisten zur Herstellung eines Medikaments zur Behandlung von pathologischen Zuständen, die mit der Zellmigration verbunden sein können oder durch eine spezifische Regulation der Zellmigration oder Chemotaxis behandelt werden können, wobei der pathologische Zustand ausgewählt ist aus der Liste, bestehend aus Arteriosklerose, Anglogenese, Wundheilung, einschließlich der Neubildung von Gefäßwänden, Verbrennungen und Entzündung, wobei der Gewebefaktoragonist eine Signalübertragung durch direktes Binden an TF (tissue factor; Gewebefaktor) einschließt und wobei es sich bei dem Gewebefaktoragonisten um FVII oder FVIIa oder eine Kombination davon handelt.
  2. Verwendung nach Anspruch 1, wobei es sich bei der Zelle um eine Gewebefaktor exprimierende menschliche Zelle, einschließlich Fibroblasten, Glattmuskelzellen, Tumorzellen, hämatopoietische Zellen, Monozyten, Makrophagen und Epithelialzellen handelt.
  3. Verwendung nach Anspruch 2, wobei die Zelle ferner PDGF und PDGF-Rezeptoren, insbesondere PDGF-beta-Rezeptoren exprimiert.
  4. Gewebefaktoragonist zur Herstellung eines Medikaments zur Behandlung von pathologischen Zuständen, die mit der Zellmigration verbunden sein können oder durch eine spezifische Regulation der Zellmigration oder Chemotaxis behandelt werden können, wobei der pathologische Zustand ausgewählt ist aus der Liste, bestehend aus Arteriosklerose, Anglogenese, Wundheilung, einschließlich der Neubildung von Gefäßwänden, Verbrennungen und Entzündung, wobei der Gewebefaktoragonist eine Signalübertragung durch direktes Binden an TF einschließt und wobei es sich bei dem Gewebefaktoragonisten um FVII oder FVIIa oder eine Kombination davon handelt.
  5. Gewebefaktoragonist nach Anspruch 4, wobei es sich bei der Zelle um eine Gewebefaktor exprimierende menschliche Zelle, einschließlich Fibroblasten, Glattmuskelzellen, Tumorzellen, hämatopoietische Zellen, Monozyten, Makrophagen und Epithelialzellen handelt.
  6. Gewebefaktoragonist nach Anspruch 5, wobei die Zelle ferner PDGF und PDGF-Rezeptoren, insbesondere PDGF-beta-Rezeptoren exprimiert.
  7. Verfahren zur Modulation oder Regulation der Zellmigration in vitro in einer Gewebefaktor exprimierenden menschlichen Zelle oder einem ebensolchen Gewebe, wobei das Verfahren den Schritt des Inkontaktbringens der Zelle oder des Gewebes mit einem Gewebefaktoragonisten oder einem Gewebefaktorantagonisten umfasst.
  8. Verfahren nach Anspruch 7, wobei es sich bei dem Gewebefaktoragonisten um FVII oder FVIIa oder eine Kombination davon handelt
  9. Verfahren nach Anspruch 7, wobei es sich bei dem Gewebefaktorantagonisten um modifizierten FVII handelt.
  10. Verfahren nach Anspruch 7, wobei es sich bei der Zelle um eine Gewebefaktor exprimierende menschliche Zelle, einschließlich Fibroblasten, Glattmuskelzellen, Tumorzellen, hämatopoietische Zellen, Monozyten, Makrophagen und Epithelialzellen handelt.
  11. Verfahren zum Ermitteln von Arzneistoffkandidaten, ausgewählt aus Gewebefaktoragonisten und Gewebefaktorantagonisten, die die Zellmigration regulieren, wobei das Verfahren umfasst:
    a) Kultivieren einer TF exprimierenden Zelle;
    b) Messen der Migration der Zelle;
    c) Inkubieren der Zelle mit einem Arzneistoffkandidaten und
    d) Messen der Migration der inkubierten Zelle und Bestimmen von jeglicher Veränderung Im Migrationsgrad im Vergleich mit der in Schritt b gemessenen Migration, wobei eine derartige Veränderung für einen biologisch wirksamen Arzneistoffkandidaten in der Zelle hinweisend ist.
EP00943706A 1999-07-14 2000-07-14 VERWENDUNG VON FVIIa ODER EINES GEWEBEFAKTORAGONISTEN ZUR REGULIERUNG DER GENEXPRESSION UND ZELLMIGRATION ODER CHEMOTAXIS Expired - Lifetime EP1200116B1 (de)

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JP2003525028A (ja) 2003-08-26
BR0012408A (pt) 2002-03-12
CZ200240A3 (cs) 2003-04-16
WO2001005353A3 (en) 2001-07-19
ATE411041T1 (de) 2008-10-15
RU2268744C2 (ru) 2006-01-27
MXPA02000468A (es) 2008-10-06
PL353035A1 (en) 2003-10-06
US20020193302A1 (en) 2002-12-19
NO20020130L (no) 2002-03-13
CA2378249A1 (en) 2001-01-25
US20050239708A1 (en) 2005-10-27
IL147294A0 (en) 2002-08-14
KR100821644B1 (ko) 2008-04-11
HUP0302052A2 (hu) 2003-09-29
ES2316372T3 (es) 2009-04-16
KR20020025194A (ko) 2002-04-03
US20090036378A1 (en) 2009-02-05
AU5807500A (en) 2001-02-05
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HUP0302052A3 (en) 2005-12-28
US7829529B2 (en) 2010-11-09

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